Plants and seeds of spring canola variety SCV453784

ABSTRACT

The invention relates to a novel canola line designated as SCV453784. The invention also relates to the seeds, the plants, and the plant parts of canola line SCV453784 as well as to methods for producing a canola plant produced by crossing canola line SCV453784 with itself or with another canola line. The invention also relates to methods for producing a canola plant containing in its genetic material one or more transgenes and to the transgenic canola plants and plant parts produced by those methods. The invention further relates to canola lines or breeding lines and plant parts derived from canola line SCV453784, to methods for producing other canola lines or plant parts derived from canola line SCV453784 and to the canola plants, varieties, and their parts derived from use of those methods. The invention additionally relates to hybrid canola seeds, plants, and plant parts produced by crossing the line SCV453784 with another canola line.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a new and distinctive canola line,designated SCV453784.

SUMMARY OF THE INVENTION

The present invention is directed, in an embodiment, to a plant, plantpart, or seed of canola line SCV453784, representative sample of seed ofwhich was deposited under ATCC Accession No. PTA-10627. The invention isalso directed, in an embodiment, to a method for producing a canola seedcomprising crossing two canola plants and harvesting the resultantcanola seed, wherein at least one of the two canola plants is of canolaline SCV453784. In another embodiment, the invention is also directed toa method for producing a canola plant with a particular trait, such asmale sterility, herbicide or insect resistance or tolerance, or modifiedfatty acid metabolism or modified carbohydrate metabolism, wherein themethod comprises transforming the canola plant of canola line SCV453784with a nucleic acid molecule that confers that trait.

DEFINITIONS

-   Allele. Allele is any of one or more alternative forms of a gene    which relate to one trait or characteristic. In a diploid cell or    organism, the two alleles of a given gene occupy corresponding loci    on a pair of homologous chromosomes.-   Alter. The utilization of up-regulation, down-regulation, or gene    silencing.-   Anther arrangement. The orientation of the anthers in fully opened    flowers; anther arrangement can be useful as an identifying trait.    Anther arrangements can range from introse (facing inward toward    pistil), erect (neither inward not outward), or extrose (facing    outward away from pistil).-   Anther dotting. The presence/absence of anther dotting (colored    spots on the tips of anthers); if present, the percentage of anther    dotting on the tips of anthers in newly opened flowers is a    distinguishing trait for varieties.-   Anther fertility. A measure of the amount of pollen produced on the    anthers of a flower. Anther fertility can range from sterile (such    as in female parents used for hybrid seed production) to fertile    (all anthers shedding).-   Backcrossing. A process in which a breeder repeatedly crosses hybrid    progeny back to one of the parents; for example, a first generation    hybrid F₁ may be crossed with one of the parental genotypes of the    F₁ hybrid.-   Blackleg (Leptosphaeria maculans). A fungal canker or dry rot    disease of the actively growing crop that causes stem girdling and    lodging. In heavily infested crops, up to 100% of the stems may be    infected, resulting in major yield loss. For purposes of this    application, resistance to blackleg is measured using ratings of “R”    (resistant), “MR” (moderately resistant), “MS” (moderately    susceptible) or “S” (susceptible).-   Cell. As used herein, the term cell includes a plant cell, whether    isolated, in tissue culture, or incorporated in a plant or plant    part.-   Cotyledon width. The cotyledons are leaf structures that form in the    developing seeds of canola that make up the majority of the mature    seed of these species. When the seed germinates, the cotyledons are    pushed out of the soil by the growing hypocotyls (segment of the    seedling stem below the cotyledons and above the root) and they    unfold as the first photosynthetic leafs of the plant. The width of    the cotyledons varies by variety and can be classified as narrow,    medium, or wide.-   Elite canola line. A canola line which has been sold commercially.-   Elite canola parent line. A canola line which is the parent line of    a canola line which has been commercially sold.-   Embryo. The embryo is the small plant contained within a mature    seed.-   Essentially all of the physiological and morphological    characteristics. This phrase refers to a plant having essentially    all of the physiological and morphological characteristics of the    recurrent parent, except for the characteristics derived from any    converted trait.-   Fatty Acid Methyl Ester (“FAME”) analysis. A method that allows for    accurate quantification of the fatty acids which make up complex    lipid classes.-   Flower bud location. The location of the unopened flower buds    relative to the adjacent opened flowers is useful in distinguishing    between the canola species. The unopened buds are held above the    most recently opened flowers in B. napus and they are positioned    below the most recently opened flower buds in B. rapa.-   Flowering date. This is measured by the number of days from planting    to the stage when 50% of the plants in a population have one or more    open flowers. This varies from variety to variety.-   Fusarium Wilt. Fusarium wilt, largely caused by Fusarium oxysporum,    is a disease of canola that causes part or all of a plant to wilt,    reducing yield by 30% or more on badly affected fields. For purposes    of this application, resistance to Fusarium wilt is measured using    ratings of “R” (resistant), “MR” (moderately resistant), “MS”    (moderately susceptible) or “S” (susceptible).-   Gene silencing. Gene silencing means the interruption or suppression    of the expression of a gene at the level of transcription or    translation.-   Genotype. This term refers to the genetic constitution of a cell or    organism.-   Glucosinolates. A secondary metabolite of Brassicales that is    organic and contains sulfer and nitrogen; derived from glucose and    an amino acid. Glucosinolates are measured in micromoles (μm) of    total alipathic glucosinolates per gram of air-dried oil-free meal.    The level of glucosinolates is somewhat influenced by the sulfur    fertility of the soil, but is also controlled by the genetic makeup    of each variety and thus can be useful in characterizing varieties.-   Growth habit. At the end of flowering, the angle relative to the    ground surface of the outermost fully expanded leaf petioles; a    variety-specific trait. This trait can range from erect (very    upright along the stem) to prostrate (almost horizontal and parallel    with the ground surface).-   Leaf attachment to the stem. The manner in which the base of the    leaf blade clasps the stem. This trait is especially useful for    distinguishing between the two canola species. The base of the leaf    blade of the upper stem leaves of B. rapa completely clasp the stem    whereas those of the B. napus only partially clasp the stem. Those    of the mustard species do not clasp the stem at all.-   Leaf blade color. The color of the leaf blades is variety-specific    and can range from light to medium dark green to blue-green.-   Leaf development of lobes. The varying degrees of development of    lobes on leaves on the upper portion of the stem which are    disconnected from one another along the petiole of the leaf. The    degree of lobing is variety-specific and can range from absent (no    lobes)/weak to very strong (abundant lobes).-   Leaf glaucosity. This refers to the waxiness of the leaves and is    characteristic of specific varieties although environment can have    some effect on the degree of waxiness. This trait can range from    absent (no waxiness)/weak to very strong. The degree of waxiness can    be best determined by rubbing the leaf surface and noting the degree    of wax present.-   Leaf indentation of margin. The varying degrees of serration along    the leaf margins on leaves on the upper portion of the stem. The    degree of serration or indentation of the leaf margins can vary from    absent (smooth margin)/weak to strong (heavy saw-tooth like margin).-   Leaf pubescence. The leaf pubescence is the degree of hairiness of    the leaf surface and is especially useful for distinguishing between    the canola species. There are two main classes of pubescence,    glabrous (smooth/not hairy) and pubescent (hairy), which mainly    differentiate between the B. napus and B. rapa species,    respectively.-   Leaf surface. A measure of the surface texture of a leaf; the leaf    surface can be used to distinguish between varieties. The surface    can be smooth or rugose (lumpy) with varying degrees between the two    extremes.-   Linkage. This term refers to a phenomenon wherein alleles on the    same chromosome tend to segregate together more often than expected    if their transmission was independent.-   Linkage disequilibrium. A phenomenon wherein alleles tend to remain    together in linkage groups when segregating from parents to    offspring, with a greater frequency than expected from their    individual frequencies.-   Locus. A position on a genomic sequence that is usually found by a    point of reference, for example, the position of a DNA sequence that    is a gene, or part of a gene or intergenic region. A locus confers    one or more traits such as, for example, male sterility, herbicide    tolerance, insect resistance, disease resistance, modified fatty    acid metabolism, modified phytic acid metabolism, modified    carbohydrate metabolism and modified protein metabolism. The trait    may be, for example, conferred by a naturally occurring gene    introduced into the genome of the variety by backcrossing, a natural    or induced mutation, or a transgene introduced through genetic    transformation techniques. A locus may comprise one or more alleles    integrated at a single chromosomal location.-   Lodging resistance. This term refers to a variety's ability to    remain erect. Lodging is rated on a scale of 1 to 5, wherein a score    of 1 indicates erect plants and a score of 5 indicates plants that    are lying on the ground.-   Maturity. The maturity of a variety is measured as the number of    days between planting and physiological maturity. This trait is    useful in distinguishing varieties relative to one another and when    used in this context it is referred to as “Relative Maturity”.-   Oil content. Oil content is measured as a percent of the whole dried    seed and is variety-specific. It can be determined using various    analytical techniques such as nuclear magnetic resonance (NMR)    spectroscopy, near-infrared (NIR) spectroscopy, and Soxhlet    extraction.-   Percent linolenic acid. This refers to the percent of total oil in    the seed that is linolenic acid.-   Percent oleic acid (OLE). OLE refers to the percent of total oil in    the seed that is oleic acid.-   Percentage of total fatty acids. This may be determined by    extracting a sample of oil from seed, producing the methyl esters of    fatty acids present in that oil sample and analyzing the proportions    of the various fatty acids in the sample using gas chromatography.    The fatty acid composition can also be a distinguishing    characteristic of a variety.-   Petal color. The petal color on the first day a flower opens can be    a distinguishing characteristic for a variety. It can be white,    varying shades of yellow, or orange.-   Plant. As used herein, the term “plant” includes plant cells, plant    protoplasts, plant cell tissue cultures from which canola plants can    be regenerated, plant calli, plant clumps, and plant cells that are    intact in plants or parts of plants, such as embryos, pollen,    ovules, flowers, pods, leaves, roots, root tips, anthers,    cotyledons, hypocotyls, meristematic cells, stems, pistils, petiole,    and an immature or mature whole plant, including a plant from which    seed or grain or anthers have been removed. Seed or embryo that will    produce the plant is also considered to be the plant.-   Plant height. This is the height of the plant at the end of    flowering if the floral branches are extended upright (i.e., not    lodged). This varies from variety to variety and although it can be    influenced by environment, relative comparisons between varieties    grown side by side are useful for variety identification.-   Plant parts. As used herein, the term “plant parts” (or “canola    plant, or a part thereof” or similar phrasings) includes    protoplasts, leaves, stems, roots, root tips, anthers, pistils,    seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, pod,    flower, shoot, tissue, petiole, cells, meristematic cells and the    like.-   Protein content. Protein content is measured as a percent of whole    dried seed and may vary from variety to variety. This can be    determined using various analytical techniques such as NIR and    Kjeldahl.-   Quantitative trait loci (QTL). This term refers to genetic loci that    control to some degree numerically-representable traits that are    usually continuously distributed.-   Regeneration. Regeneration refers to the development of a plant from    tissue culture.-   Resistance to lodging. This term refers to the ability of a variety    to stand up in the field under high yield conditions and severe    environmental factors. A variety can have very good, good (remains    upright), fair, or poor (falls over) resistance to lodging. The    degree of resistance to lodging is not expressed under all    conditions but is most meaningful when there is some degree of    lodging in a field trial.-   Seed coat color. The color of the seed coat can be variety-specific    and can range from black to brown to yellow. Color can also be mixed    for some varieties.-   Seed coat mucilage. This refers to a gel-like substance on the seed    coat that draws water to itself. It can be useful for    differentiating between the two species of canola with B. rapa    varieties having mucilage present in their seed coats whereas B.    napus varieties do not have this present. It is detected by imbibing    seeds with water and monitoring the mucilage that is exuded by the    seed.-   Seedling growth habit. This term refers to the rosette, which    consists of the first 2-8 true leaves; a variety can be    characterized as having a strong rosette (closely packed leaves) or    a weak rosette (loosely arranged leaves).-   Silique (pod) habit. This term refers to the orientation of the pods    along the racemes (flowering stems) and is variety-specific. This    trait can range from erect (pods angled close to racemes) to    horizontal (pods perpendicular to racemes) to arching (pods show    distinct arching habit).-   Silique (pod) length of beak. The beak is the segment at the end of    the pod which does not contain seed (it is a remnant of the stigma    and style for the flower). The length of the beak can be    variety-specific and can range from short to medium to long.-   Silique (pod) length of pedicel. The pedicel is the stem that    attaches the pod to the raceme or flowering shoot. The length of the    pedicel can be variety-specific and can vary from short to medium to    long.-   Silique (pod) length. This is the length of the fully developed pods    and can range from short to medium to long. In characterizing a    variety, it is best used by making comparisons relative to reference    varieties.-   Silique (pod) type. The type of pod; this is typically a bilateral    single pod for both species of canola and is, therefore, not very    useful for variety identification within these species.-   Silique (pod) width. This is the width of a fully developed pod and    can range from narrow to medium to wide. In characterizing a    variety, it is best used by making comparisons relative to reference    varieties.-   Single gene converted (conversion). This term refers to plants that    are developed using a plant breeding technique known as    backcrossing, or via genetic engineering, wherein essentially all of    the desired morphological and physiological characteristics of a    variety are recovered in addition to the single gene transferred    into the variety via the backcrossing technique or via genetic    engineering.-   Stem intensity of anthocyanin coloration. The varying degrees of    purple coloration on stems and other organs of canola plants, which    is due to the presence of anthocyanin (purple) pigments. The degree    of coloration is somewhat subject to growing conditions, but    varieties typically show varying degrees of coloration ranging from:    absent (no purple) to very weak to very strong (deep purple    coloration).-   Total saturated (TOTSAT). Measured as a percent of the total oil of    the seed, this refers to the amount of saturated fats in the oil    including C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 and C24.0.

DETAILED DESCRIPTION OF THE INVENTION

Reference now will be made in detail to the embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, not alimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, can be used on another embodiment to yield a stillfurther embodiment.

Thus, it is intended that the present invention covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent invention are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only, and is not intended as limiting the broader aspects ofthe present invention.

According to the invention, there is provided a new canola linedesignated SCV453784. This invention relates to the seeds, plants, andplant parts of canola SCV453784 and to methods for producing a canolaplant produced by crossing the canola SCV453784 with itself or anothercanola genotype, and to the creation of variants by mutagenesis ortransformation of canola SCV453784.

Thus, any such methods using the canola line SCV453784 are part of thisinvention, including but not limited to selfing, backcrossing, hybridproduction, and crosses to populations. All plants produced using canolaline SCV453784 as a parent are within the scope of this invention. In anembodiment, the canola line could be used in crosses with otherdifferent canola plants to produce first generation (F₁) canola hybridseeds and plants with superior characteristics.

In another embodiment, the present invention provides for single ormultiple gene-converted plants of SCV453784. The transferred gene(s) maybe a dominant or recessive allele. The transferred gene(s) may confersuch traits as herbicide tolerance, insect resistance, resistance forbacterial, fungal, or viral disease, male fertility, male sterility,enhanced nutritional quality, modified fatty acid metabolism, modifiedcarbohydrate metabolism, modified seed yield, modified oil percent,modified protein percent, modified lodging resistance, modifiedglucosinolate content, modified chlorophyll content and industrialusage. The gene may be a naturally occurring canola gene or a transgeneintroduced through genetic engineering techniques.

In another embodiment, the present invention provides regenerable cellsfor use in tissue culture of canola plant SCV453784. The tissue culturemay be capable of regenerating plants having essentially all of thephysiological and morphological characteristics of the foregoing canolaplant, and capable of regenerating plants having substantially the samegenotype as the foregoing canola plant. The regenerable cells in suchtissue cultures may be embryos, protoplasts, meristematic cells, callus,pollen, leaves, anthers, pistils, cotyledons, roots, root tips, flowers,seeds, pods or stems. Still further, the present invention, in anembodiment, provides canola plants regenerated from the tissue culturesof the invention.

In a particular embodiment, the invention comprises a plant cell or adescendant of a plant cell of a Brassica plant designated varietySCV453784. In some embodiments, the descendant has the same desirabletraits as the plant designated variety SCV453784.

In another aspect, the present invention provides a method ofintroducing a desired trait into canola line SCV453784, wherein themethod comprises crossing a SCV453784 plant with a plant of anothercanola genotype that comprises a desired trait to produce F₁ progenyplants, wherein the desired trait is selected from the group consistingof male sterility, herbicide tolerance, insect resistance, modifiedfatty acid metabolism, modified carbohydrate metabolism, modified seedyield, modified oil percent, modified protein percent, modified lodgingresistance, and resistance to bacterial disease, fungal disease, orviral disease; selecting one or more progeny plants that have thedesired trait to produce selected progeny plants; crossing the selectedprogeny plants with the SCV453784 plants to produce backcross progenyplants; selecting for backcross progeny plants that have the desiredtrait and essentially all of the physiological and morphologicalcharacteristics of canola line SCV453784 to produce selected backcrossprogeny plants; and repeating these steps three or more times to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and essentially all of the physiological and morphologicalcharacteristics of canola line SCV453784 as listed in Table 1. Includedin this aspect of the invention is the plant produced by the methodwherein the plant has the desired trait and essentially all of thephysiological and morphological characteristics of canola line SCV453784as listed in Table 1.

A. Origin and Breeding History

Spring canola variety SCV453784 is a spring canola inbred line developedfrom the cross of SCV425044 and SCV378221 (proprietary spring canolainbred lines of Monsanto Technology LLC) which was self pollinated andthe pedigree system of plant breeding was then used to developSCV453784. Some of the criteria used for selection in variousgenerations include: fertility, standability, disease tolerance,combining ability, oil content, maturity and total saturated fats.

SCV453784 is a conventional (non transgenic), pollen-fertile canolarestorer inbred line (R-line) used for producing hybrids with resistanceto blackleg and Fusarium wilt diseases. Canola line SCV453784 is stable,uniform and no off-type plants have been exhibited in evaluation. Theline has shown uniformity and stability, as described in the followingvariety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity.

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a canola plant having the physiological and morphologicalcharacteristics of canola variety SCV453784. A description ofphysiological and morphological characteristics of canola varietySCV453784 is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of SCV453784VALUE CHARACTERISTIC SCV453784 SCV378221* SCV204738* 1. PLANT PlantHeight 140 cm 125 cm 130 cm Days to 50% 50 days 49 days 50 daysFlowering Maturity Medium Early Early Resistance To Good Good Very GoodLodging Blackleg Resistance MR MR R Fusarium Wilt R S R Resistance RfRestorer Gene From “old” From “old” From “old” Source source sourcesource (INRA) (INRA) (INRA) 2. SEED Coat Color Black Black Black OilContent 44.08% 45.82% 45.68% (% of whole seed @ 8.5% moisture)** ProteinContent (% 48.96% 46.87% 45.86% defatted, dry meal)** Erucic AcidContent Trace Trace Trace Glucosinolate 16.77% 16.23% 16.38% Content***SCV378221 and SCV204738 are proprietary canola inbred lines of MonsantoTechnology LLC. **These are average values. Values may vary due toenvironment. Other values that are substantially equivalent are alsowithin the scope of the invention.

In an embodiment, the invention is directed to methods for producing acanola plant by crossing a first parent canola plant with a secondparent canola plant, wherein the first or second canola plant is thecanola plant from the line SCV453784. Further, both first and secondparent canola plants may be from the line SCV453784. Any methods usingthe line SCV453784 are part of this invention: selfing, backcrosses,hybrid breeding, and crosses to populations. Any plants produced usingas a parent line SCV453784 are within the scope of the invention.

Additional methods of the present invention include, but are not limitedto, expression vectors introduced into plant tissues using a direct genetransfer method such as microprojectile-mediated delivery, DNAinjection, and electroporation. Expression vectors may be introducedinto plant tissues by using either microprojectile-mediated deliverywith a biolistic device or by using Agrobacterium-mediatedtransformation. Transformant plants obtained with the protoplasm of theinvention are intended to be within the scope of this invention.

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies which are inserted into the genome using transformation, arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transgenic variant of SCV453784 may contain at leastone transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10transgenes and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, or 2 transgenes. Over the last fifteen to twenty years severalmethods for producing transgenic plants have been developed, and thepresent invention also relates to transgenic variants of the claimedcanola line SCV453784.

One embodiment of the invention is a process for producing canola lineSCV453784 further comprising a desired trait, said process comprisingtransforming a canola plant of line SCV453784 with a transgene thatconfers a desired trait. Another embodiment of the invention comprises acanola plant produced by this process. In one embodiment, the desiredtrait may be one or more of herbicide tolerance, insect resistance,disease resistance, modified seed yield, modified oil percent, modifiedprotein percent, modified lodging resistance, or modified fatty acid orcarbohydrate metabolism. The specific gene may be any gene known in theart or listed herein, including but not limited to a polynucleotideconferring resistance or tolerance to imidazolinone, sulfonylurea,glyphosate, glufosinate, 2,4-D, Dicamba, L-phosphinothricin, triazine,hydroxyphenylpyruvate dioxygenase inhibitor, protoporphyrinogen oxidaseinhibitor, phenoxy proprionic acid, cyclohexone, and benzonitrile; apolynucleotide encoding a Bacillus thuringiensis polypeptide, apolynucleotide encoding phytase, fatty acid desaturase (FAD)-2, FAD-3,galactinol synthase or a raffinose synthetic enzyme; or a polynucleotideconferring resistance to blackleg, white rust or other common canoladiseases.

Numerous methods for plant transformation have been developed and areincluded as part of the invention, including biological and physicalplant transformation protocols. See, for example, Miki, et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88 and “GeneticTransformation for the Improvement of Canola”, in World Conference onBiotechnology for the Fats and Oils Industry. 1988. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available and are includedwithin the invention. See e.g. Gruber, et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

As an embodiment of the invention, a genetic trait which has beenengineered into the genome of a particular canola plant may then bemoved into the genome of another variety using traditional breedingtechniques that are well-known in the plant breeding arts. For example,a backcrossing approach may be used to move a transgene from atransformed canola variety into an already-developed canola variety, andthe resulting backcross conversion plant would then comprise thetransgene(s).

As an embodiment of the invention, various genetic elements can beintroduced into the plant genome using transformation techniques. Theseelements include, but are not limited to genes, coding sequences,inducible, constitutive, and tissue specific promoters, enhancingsequences, and signal and targeting sequences. For example, see thetraits, genes and transformation methods listed in U.S. Pat. No.6,118,055, incorporated herein by reference in its entirety.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed canola plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the canola plant(s).

Expression Vectors for Canola Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Any selectable markerknown in the art and/or discussed herein may be used in the presentinvention. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley, et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferaseand the bleomycin resistance determinant. Hayford, et al., PlantPhysiol. 86:1216 (1988), Jones, et al., Mol. Gen. Genet., 210:86 (1987),Svab, et al., Plant Mol. Biol. 14:197 (1990) Hille, et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance ortolerance to herbicides such as glyphosate, glufosinate or bromoxynil.Comai, et al., Nature 317:741-744 (1985), Gordon-Kamm, et al., PlantCell 2:603-618 (1990) and Stalker, et al., Science 242:419-423 (1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz, et al., Somatic Cell Mol. Genet. 13:67 (1987),Shah, et al., Science 233:478 (1986), Charest, et al., Plant Cell Rep.8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri, et al.,EMBO J. 8:343 (1989), Koncz, et al., Proc. Natl. Acad. Sci. U.S.A.84:131 (1987), DeBlock, et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN J., p. 1-4 (1993) and Naleway, et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Canola Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter. Anypromoter known in the art and/or discussed herein may be used in thepresent invention. Several types of promoters are now well-known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

-   -   A. Inducible Promoters—An inducible promoter is operably linked        to a gene for expression in canola. Optionally, the inducible        promoter is operably linked to a nucleotide sequence encoding a        signal sequence which is operably linked to a gene for        expression in canola. With an inducible promoter the rate of        transcription increases in response to an inducing agent.    -   Any inducible promoter can be used in the instant invention. See        Ward, et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary        inducible promoters include, but are not limited to, that from        the activating copper-metallothionein expression I (ACEI) system        which responds to copper (Mett, et al., PNAS 90:4567-4571        (1993)); In2 gene from maize which responds to        benzenesulfonamide herbicide safeners (Hershey, et al., Mol.        Gen. Genetics 227:229-237 (1991) and Gatz, et al., Mol. Gen.        Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, et        al., Mol. Gen. Genetics 227:229-237 (1991)). A particular        inducible promoter responds to an inducing agent to which plants        do not normally respond. An exemplary inducible promoter is the        inducible promoter from a steroid hormone gene, the        transcriptional activity of which is induced by a        glucocorticosteroid hormone. Schena, et al., Proc. Natl. Acad.        Sci. U.S.A. 88:0421 (1991).    -   B. Constitutive Promoters—A constitutive promoter is operably        linked to a gene for expression in canola or the constitutive        promoter is operably linked to a nucleotide sequence encoding a        signal sequence which is operably linked to a gene for        expression in canola.    -   Many different constitutive promoters can be utilized in the        instant invention. Exemplary constitutive promoters include, but        are not limited to, the promoters from plant viruses such as the        35S promoter from CaMV (Odell, et al., Nature 313:810-812        (1985)) and the promoters from such genes as rice actin        (McElroy, et al., Plant Cell 2:163-171 (1990)); ubiquitin        (Christensen, et al., Plant Mol. Biol. 12:619-632 (1989) and        Christensen, et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU        (Last, et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS        (Velten, et al., EMBO J. 3:2723-2730 (1984)) and maize H3        histone (Lepetit, et al., Mol. Gen. Genetics 231:276-285 (1992)        and Atanassova, et al., Plant Journal 2 (3): 291-300 (1992)).        The ALS promoter, Xbal/Ncol fragment 5′ to the Brassica napus        ALS3 structural gene (or a nucleotide sequence similarity to        said Xbal/Ncol fragment), represents a particularly useful        constitutive promoter. See PCT Application No. WO 96/30530,        incorporated herein by reference in its entirety.    -   C. Tissue-Specific or Tissue-Preferred Promoters—A        tissue-specific promoter is operably linked to a gene for        expression in canola. Optionally, the tissue-specific promoter        is operably linked to a nucleotide sequence encoding a signal        sequence which is operably linked to a gene for expression in        canola. Plants transformed with a gene of interest operably        linked to a tissue-specific promoter produce the protein product        of the transgene exclusively, or preferentially, in a specific        tissue.    -   Any tissue-specific or tissue-preferred promoter can be utilized        in the instant invention. Exemplary tissue-specific or        tissue-preferred promoters include, but are not limited to, a        root-preferred promoter such as that from the phaseolin gene        (Murai, et al., Science 23:476-482 (1983) and Sengupta-Gopalan,        et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a        leaf-specific and light-induced promoter such as that from cab        or rubisco (Simpson, et al., EMBO J. 4(11):2723-2729 (1985) and        Timko, et al., Nature 318:579-582 (1985)); an anther-specific        promoter such as that from LAT52 (Twell, et al., Mol. Gen.        Genetics 217:240-245 (1989)); a pollen-specific promoter such as        that from Zm13 (Guerrero, et al., Mol. Gen. Genetics 244:161-168        (1993)) or a microspore-preferred promoter such as that from apg        (Twell, et al., Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See e.g.Becker, et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al., PlantMol. Biol. 9:3-17 (1987), Lerner, et al., Plant Physiol. 91:124-129(1989), Fontes, et al., Plant Cell 3:483-496 (1991), Matsuoka, et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould, et al., J. Cell. Biol.108:1657 (1989), Creissen, et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Steifel, et al., Plant Cell 2:785-793(1990). Any signal sequence known in the art and/or discussed herein maybe used in the present invention.

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to an embodiment, the transgenic plant provided for commercialproduction of foreign protein is a canola plant. In another embodiment,the biomass of interest is seed. For the relatively small number oftransgenic plants that show higher levels of expression, a genetic mapcan be generated, primarily via conventional restriction fragment lengthpolymorphism (RFLP), polymerase chain reaction (PCR), short sequencerepeats (SSR) analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Wang, et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are becoming increasinglyavailable for the canola genome. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. Single nucleotide polymorphism (SNP)s may alsobe used alone or in combination with other techniques.

In an embodiment of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of canola the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide tolerance,agronomic, grain quality and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to canola as well as non-native DNAsequences can be transformed into canola and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into thegenome for the purpose of altering the expression of proteins. Reductionof the activity of specific genes (also known as gene silencing, or genesuppression) may also desirable for several aspects of geneticengineering in plants.

Many techniques for gene silencing are well-known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118, Springer-Verlag, 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy, et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829, incorporated herein by reference intheir entirety); co-suppression (e.g., Taylor (1997) Plant Cell 9:1245;Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA91:3490-3496; Finnegan, et al. (1994) Bio/Technology 12: 883-888; andNeuhuber, et al. (1994) Mol. Gen. Genet. 244:230-241); RNA interference(Napoli, et al. (1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323,incorporated herein by reference in its entirety; Sharp (1999) GenesDev. 13:139-141; Zamore, et al. (2000) Cell 101:25-33; and Montgomery,et al. (1998) PNAS USA 95:15502-15507), virus-induced gene silencing(Burton, et al. (2000) Plant Cell 12:691-705; and Baulcombe (1999) Curr.Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff, etal. (1988) Nature 334: 585-591); hairpin structures (Smith, et al.(2000) Nature 407:319-320; WO 99/53050; and WO 98/53083), incorporatedherein by reference in their entirety; MicroRNA (Aukerman & Sakai (2003)Plant Cell 15:2730-2741); ribozymes (Steinecke, et al. (1992) EMBO J.11:1525; and Perriman, et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853, incorporated herein by reference in their entirety);Zn-finger targeted molecules (e.g., WO 01/52620; WO 03/048345; and WO00/42219, incorporated herein by reference in their entirety); and othermethods or combinations of the above methods known to those of skill inthe art.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

-   -   1. Genes That Confer Resistance to Pests or Disease and That        Encode:        -   A. Plant disease resistance genes. Plant defenses are often            activated by specific interaction between the product of a            disease resistance gene (R) in the plant and the product of            a corresponding avirulence (Avr) gene in the pathogen. A            plant variety can be transformed with cloned resistance            genes to engineer plants that are resistant to specific            pathogen strains. See, for example Jones, et al., Science            266:789 (1994) (cloning of the tomato Cf-9 gene for            resistance to Cladosporium fulvum); Martin, et al., Science            262:1432 (1993) (tomato Pto gene for resistance to            Pseudomonas syringae pv. tomato encodes a protein kinase);            Mindrinos, et al., Cell 78:1089 (1994) (Arabidopsis RSP2            gene for resistance to Pseudomonas syringae), McDowell &            Woffenden, (2003) Trends Biotechnol. 21(4): 178-83 and            Toyoda, et al., (2002) Transgenic Res. 11 (6):567-82.        -   B. A gene conferring resistance to fungal pathogens, such as            oxalate oxidase or oxalate decarboxylase (Zhou, et            al., (1998) Pl. Physiol. 117:33-41).        -   C. A Bacillus thuringiensis protein, a derivative thereof or            a synthetic polypeptide modeled thereon. See, for example,            Geiser, et al., Gene 48:109 (1986), who disclose the cloning            and nucleotide sequence of a Bt δ-endotoxin gene. Moreover,            DNA molecules encoding δ-endotoxin genes can be purchased            from American Type Culture Collection, Manassas, Va., for            example, under ATCC Accession Nos. 40098, 67136, 31995 and            31998.        -   D. A lectin. See, for example, the disclosure by Van Damme,            et al., Plant Molec. Biol. 24:25 (1994), who disclose the            nucleotide sequences of several Clivia miniata            mannose-binding lectin genes.        -   E. A vitamin-binding protein such as avidin. See PCT            Application No. WO 93/06487, incorporated herein by            reference in its entirety. The application teaches the use            of avidin and avidin homologues as larvicides against insect            pests.        -   F. An enzyme inhibitor, for example, a protease or            proteinase inhibitor or an amylase inhibitor. See, for            example, Abe, et al., J. Biol. Chem. 262:16793 (1987)            (nucleotide sequence of rice cysteine proteinase inhibitor),            Huub, et al., Plant Molec. Biol. 21:985 (1993) (nucleotide            sequence of cDNA encoding tobacco proteinase inhibitor I),            Sumitani, et al., Biosci. Biotech. Biochem. 57:1243 (1993)            (nucleotide sequence of Streptomyces nitrosporeus α-amylase            inhibitor) and U.S. Pat. No. 5,494,813 (Hepher and Atkinson,            issued Feb. 27, 1996), incorporated herein by reference in            its entirety.        -   G. An insect-specific hormone or pheromone such as an            ecdysteroid or juvenile hormone, a variant thereof, a            mimetic based thereon, or an antagonist or agonist thereof.            See, for example, the disclosure by Hammock, et al., Nature            344:458 (1990), of baculovirus expression of cloned juvenile            hormone esterase, an inactivator of juvenile hormone.        -   H. An insect-specific peptide or neuropeptide which, upon            expression, disrupts the physiology of the affected pest.            For example, see the disclosures of Regan, J Biol. Chem.            269:9 (1994) (expression cloning yields DNA coding for            insect diuretic hormone receptor), and Pratt, et al.,            Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin            is identified in Diploptera puntata). See also U.S. Pat. No.            5,266,317, incorporated herein by reference in its entirety,            to Tomalski, et al., who disclose genes encoding            insect-specific, paralytic neurotoxins.        -   I. An insect-specific venom produced in nature by a snake, a            wasp, etc. For example, see Pang et al., Gene 116:165            (1992), for disclosure of heterologous expression in plants            of a gene coding for a scorpion insectotoxic peptide.        -   J. An enzyme responsible for a hyperaccumulation of a            monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a            phenylpropanoid derivative or another non-protein molecule            with insecticidal activity.        -   K. An enzyme involved in the modification, including the            post-translational modification, of a biologically active            molecule; for example, a glycolytic enzyme, a proteolytic            enzyme, a lipolytic enzyme, a nuclease, a cyclase, a            transaminase, an esterase, a hydrolase, a phosphatase, a            kinase, a phosphorylase, a polymerase, an elastase, a            chitinase and a glucanase, whether natural or synthetic. See            PCT Application No. WO 93/02197, incorporated herein by            reference in its entirety, in the name of Scott, et al.,            which discloses the nucleotide sequence of a callase gene.            DNA molecules which contain chitinase-encoding sequences can            be obtained, for example, from the ATCC under Accession Nos.            39637 and 67152. See also Kramer, et al., Insect Biochem.            Molec. Biol. 23:691 (1993), who teach the nucleotide            sequence of a cDNA encoding tobacco hornworm chitinase, and            Kawalleck, et al., Plant Molec. Biol. 21:673 (1993), who            provide the nucleotide sequence of the parsley ubi4-2            polyubiquitin gene, U.S. Pat. Nos. 7,145,060, 7,087,810 and            6,563,020, incorporated herein by reference in their            entirety.        -   L. A molecule that stimulates signal transduction. For            example, see the disclosure by Botella, et al., Plant Molec.            Biol. 24:757 (1994), of nucleotide sequences for mung bean            calmodulin cDNA clones, and Griess, et al., Plant Physiol.            104:1467 (1994), who provide the nucleotide sequence of a            maize calmodulin cDNA clone.        -   M. A hydrophobic moment peptide. See PCT Application No. WO            95/16776 (disclosure of peptide derivatives of Tachyplesin            which inhibit fungal plant pathogens) and PCT application WO            95/18855 and U.S. Pat. No. 5,607,914 which teaches synthetic            antimicrobial peptides that confer disease resistance, each            of which is incorporated herein by reference in its            entirety.        -   N. A membrane permease, a channel former or a channel            blocker. For example, see the disclosure of Jaynes, et al.,            Plant Sci 89:43 (1993), of heterologous expression of a            cecropin-β, lytic peptide analog to render transgenic            tobacco plants resistant to Pseudomonas solanacearum.        -   O. A viral-invasive protein or a complex toxin derived            therefrom. For example, the accumulation of viral coat            proteins in transformed plant cells imparts resistance to            viral infection and/or disease development effected by the            virus from which the coat protein gene is derived, as well            as by related viruses. See Beachy, et al., Ann. rev.            Phytopathol. 28:451 (1990). Coat protein-mediated resistance            has been conferred upon transformed plants against alfalfa            mosaic virus, cucumber mosaic virus, tobacco streak virus,            potato virus X, potato virus Y, tobacco etch virus, tobacco            rattle virus and tobacco mosaic virus. Id.        -   P. An insect-specific antibody or an immunotoxin derived            therefrom. Thus, an antibody targeted to a critical            metabolic function in the insect gut would inactivate an            affected enzyme, killing the insect. Cf. Taylor, et al.,            Abstract #497, Seventh Int'l Symposium on Molecular            Plant-Microbe Interactions (Edinburgh, Scotland) (1994)            (enzymatic inactivation in transgenic tobacco via production            of single-chain antibody fragments).        -   Q. A virus-specific antibody. See, for example, Tavladoraki,            et al., Nature 366:469 (1993), who show that transgenic            plants expressing recombinant antibody genes are protected            from virus attack.        -   R. A developmental-arrestive protein produced in nature by a            pathogen or a parasite. Thus, fungal            endo-α-1,4-D-polygalacturonases facilitate fungal            colonization and plant nutrient release by solubilizing            plant cell wall homo-α-1,4-D-galacturonase. See Lamb, et            al., Bio/Technology 10:1436 (1992). The cloning and            characterization of a gene which encodes a bean            endopolygalacturonase-inhibiting protein is described by            Toubart, et al., Plant J. 2:367 (1992).        -   S. A developmental-arrestive protein produced in nature by a            plant. For example, Logemann, et al., Bio/Technology 10:305            (1992), have shown that transgenic plants expressing the            barley ribosome-inactivating gene have an increased            resistance to fungal disease.        -   T. Genes involved in the Systemic Acquired Resistance (SAR)            Response and/or the pathogenesis-related genes. Briggs, S.,            Current Biology, 5(2) (1995); Pieterse & Van Loon (2004)            Curr. Opin. Plant Bio. 7(4):456-64 and Somssich (2003) Cell            113(7):815-6.        -   U. Antifungal genes. See Cornelissen and Melchers, Plant            Physiol., 101:709-712 (1993); Parijs, et al., Planta            183:258-264 (1991) and Bushnell, et al., Can. J. of Plant            Path. 20(2):137-149 (1998); see also U.S. Pat. No.            6,875,907, incorporated herein by reference in its entirety.        -   V. Detoxification genes, such as for fumonisin, beauvericin,            moniliformin and zearalenone and their structurally related            derivatives. For example, see U.S. Pat. No. 5,792,931,            incorporated herein by reference in its entirety.        -   W. Cystatin and cysteine proteinase inhibitors. See U.S.            Pat. No. 7,205,453, incorporated herein by reference in its            entirety.        -   X. Defensin genes. See WO 03/000863 and U.S. Pat. No.            6,911,577, incorporated herein by reference in their            entirety.        -   Y. Genes that confer resistance to Phytophthora root rot,            such as the Brassica equivalents of the Rps 1, Rps 1-a, Rps            1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps            3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps            genes. See, for example, Shoemaker, et al., Phytophthora            Root Rot Resistance Gene Mapping in Soybean, Plant Genome IV            Conference, San Diego, Calif. (1995).    -   2. Genes That Confer Resistance or Tolerance to an Herbicide:        -   A. An herbicide that inhibits the growing point or meristem,            such as an imidazolinone or a sulfonylurea. Exemplary genes            in this category code for mutant ALS and AHAS enzyme as            described, for example, by Lee, et al., EMBO J. 7:1241            (1988), and Miki, et al., Theor. Appl. Genet. 80:449 (1990),            respectively.        -   B. Glyphosate (resistance or tolerance conferred by mutant            5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA            genes, respectively) and other phosphono compounds such as            glufosinate (phosphinothricin acetyl transferase (PAT) and            Streptomyces hygroscopicus PAT bar genes), and pyridinoxy or            phenoxy proprionic acids and cyclohexones (ACCase            inhibitor-encoding genes). See, for example, U.S. Pat. No.            4,940,835 to Shah, et al., incorporated herein by reference            in its entirety, which discloses the nucleotide sequence of            a form of EPSP which can confer glyphosate resistance or            tolerance. U.S. Pat. No. 5,627,061 to Barry, et al.,            incorporated herein by reference in its entirety, also            describes genes encoding EPSPS enzymes. See also U.S. Pat.            Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;            5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910;            5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;            5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.            36,449; RE 37,287 E; and 5,491,288; and international            publications EP1173580; WO 01/66704; EP1173581 and            EP1173582, which are each incorporated herein by reference            in their entireties. Glyphosate resistance or tolerance is            also imparted to plants that express a gene that encodes a            glyphosate oxido-reductase enzyme as described more fully in            U.S. Pat. Nos. 5,776,760 and 5,463,175, which are            incorporated herein by reference in their entireties. In            addition glyphosate resistance or tolerance can be imparted            to plants by the overexpression of genes encoding glyphosate            N-acetyltransferase. See, for example, U.S. Pat. No.            7,462,481, incorporated herein by reference in its entirety.            A DNA molecule encoding a mutant aroA gene can be obtained            under ATCC accession number 39256, and the nucleotide            sequence of the mutant gene is disclosed in U.S. Pat. No.            4,769,061 to Comai, incorporated herein by reference in its            entirety. European patent application No. 0 333 033 to            Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et            al., incorporated herein by reference in their entireties,            disclose nucleotide sequences of glutamine synthetase genes            which confer resistance or tolerance to herbicides such as            L-phosphinothricin.        -   The nucleotide sequence of a PAT gene is provided in            European application No. 0 242 246 to Leemans, et al.,            incorporated herein by reference in its entirety, DeGreef,            et al., Bio/Technology 7:61 (1989), describe the production            of transgenic plants that express chimeric bar genes coding            for PAT activity. Exemplary of genes conferring resistance            or tolerance to phenoxy proprionic acids and cyclohexones,            such as sethoxydim and haloxyfop are the Accl-S1, Accl-S2            and Accl-S3 genes described by Marshall, et al., Theor.            Appl. Genet. 83:435 (1992).        -   C. An herbicide that inhibits photosynthesis, such as a            triazine (psbA and gs+ genes) and a benzonitrile (nitrilase            gene). Przibila, et al., Plant Cell 3:169 (1991), describe            the transformation of Chlamydomonas with plasmids encoding            mutant psbA genes. Nucleotide sequences for nitrilase genes            are disclosed in U.S. Pat. No. 4,810,648 to Stalker,            incorporated herein by reference in its entirety, and DNA            molecules containing these genes are available under ATCC            Accession Nos. 53435, 67441, and 67442. Cloning and            expression of DNA coding for a glutathione S-transferase is            described by Hayes, et al., Biochem. J. 285:173 (1992).        -   D. Acetohydroxy acid synthase, which has been found to make            plants that express this enzyme resistant to multiple types            of herbicides, has been introduced into a variety of plants.            See Hattori, et al., Mol. Gen. Genet. 246:419, 1995. Other            genes that confer tolerance to herbicides include a gene            encoding a chimeric protein of rat cytochrome P4507A1 and            yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al.,            Plant Physiol., 106:17, 1994), genes for glutathione            reductase and superoxide dismutase (Aono, et al., Plant Cell            Physiol. 36:1687, 1995), and genes for various            phosphotransferases (Datta, et al., Plant Mol. Biol. 20:619,            1992).        -   E. Protoporphyrinogen oxidase (protox) is necessary for the            production of chlorophyll, which is necessary for all plant            survival. The protox enzyme serves as the target for a            variety of herbicidal compounds. These herbicides also            inhibit growth of all the different species of plants            present, causing their total destruction. The development of            plants containing altered protox activity which are            resistant to these herbicides are described in U.S. Pat.            Nos. 6,288,306; 6,282,837; 5,767,373; and international            publication WO 01/12825, each of which is incorporated            herein by reference in its entirety.    -   3. Genes That Confer or Contribute to a Value-Added Trait:        -   A. Modified fatty acid metabolism, for example, by            transforming a plant with an antisense gene of stearyl-ACP            desaturase to increase stearic acid content of the plant.            See Knultzon, et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624            (1992).        -   B. Decreased phytate content: 1) Introduction of a            phytase-encoding gene would enhance breakdown of phytate,            adding more free phosphate to the transformed plant. For            example, see Van Hartingsveldt, et al., Gene 127:87 (1993),            for a disclosure of the nucleotide sequence of an            Aspergillus niger phytase gene. 2) A gene could be            introduced that reduced phytate content. In maize for            example, this could be accomplished by cloning and then            reintroducing DNA associated with the single allele which is            responsible for maize mutants characterized by low levels of            phytic acid. See Raboy, et al., Maydica 35:383 (1990) and/or            by altering inositol kinase activity as in WO 02/059324,            U.S. Pat. No. 7,067,720, WO 03/027243, US2003/0079247, WO            99/05298, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224,            U.S. Pat. No. 6,391,348, WO 2002/059324, WO 98/45448, WO            99/55882, WO 01/04147, each of which is incorporated herein            by reference in its entirety.        -   C. Modified carbohydrate composition effected, for example,            by transforming plants with a gene coding for an enzyme that            alters the branching pattern of starch, or, a gene altering            thioredoxin such as NTR and/or TRX (See U.S. Pat. No.            6,531,648, which is incorporated herein by reference in its            entirety) and/or a gamma zein knock out or mutant such as            cs27 or TUSC27 or en27 (See U.S. Pat. No. 6,858,778 and U.S.            Patent Application Nos. 2005/0160488, 2005/0204418; which            are incorporated herein by reference in their entireties).            See Shiroza, et al., J. Bacteriol. 170: 810 (1988)            (nucleotide sequence of Streptococcus mutans            fructosyltransferase gene), Steinmetz, et al., Mol. Gen.            Genet. 200: 220 (1985) (nucleotide sequence of Bacillus            subtilis levansucrase gene), Pen, et al., Bio/Technology 10:            292 (1992) (production of transgenic plants that express            Bacillus licheniformis alpha-amylase), Elliot, et al., Plant            Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomato            invertase genes), Sogaard, et al., J. Biol. Chem. 268:            22480 (1993) (site-directed mutagenesis of barley            alpha-amylase gene), and Fisher, et al., Plant Physiol. 102:            1045 (1993) (maize endosperm starch branching enzyme II), WO            99/10498 (improved digestibility and/or starch extraction            through modification of UDP-D-xylose 4-epimerase, Fragile 1            and 2, Ref 1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method of            producing high oil seed by modification of starch levels            (AGP)), each of which is incorporated herein by reference in            its entirety. The fatty acid modification genes mentioned            above may also be used to affect starch content and/or            composition through the interrelationship of the starch and            oil pathways.        -   D. Elevated oleic acid via FAD-2 gene modification and/or            decreased linolenic acid via FAD-3 gene modification. See            U.S. Pat. Nos. 6,063,947; 6,323,392; and international            publication WO 93/11245, each of which is incorporated            herein by reference in its entirety.        -   E. Altering conjugated linolenic or linoleic acid content,            such as in WO 01/12800, incorporated herein by reference in            its entirety. Altering LEC1, AGP, Dek1, Superal1, mi1ps,            various Ipa genes such as Ipa1, Ipa3, hpt or hggt. For            example, see WO 02/42424, WO 98/22604, WO 03/011015, U.S.            Pat. Nos. 6,423,886, 6,197,561, 6,825,397, 7,157,621, U.S.            Patent Application No. 2003/0079247, International            publications WO 02/057439 and WO 03/011015 and            Rivera-Madrid, R., et al. Proc. Natl. Acad. Sci.            92:5620-5624 (1995), each of which is incorporated herein by            reference in its entirety.        -   F. Altered antioxidant content or composition, such as            alteration of tocopherol or tocotrienols. For example, see            U.S. Pat. Nos. 6,787,683 and 7,154,029 and WO 00/68393            involving the manipulation of antioxidant levels through            alteration of a phytl prenyl transferase (ppt), WO 03/082899            through alteration of a homogentisate geranyl geranyl            transferase (hggt), each of which is incorporated herein by            reference in its entirety.        -   G. Altered essential seed amino acids. For example, see U.S.            Pat. No. 6,127,600 (method of increasing accumulation of            essential amino acids in seeds), U.S. Pat. No. 6,080,913            (binary methods of increasing accumulation of essential            amino acids in seeds), U.S. Pat. No. 5,990,389 (high            lysine), WO 99/40209 (alteration of amino acid compositions            in seeds), WO 99/29882 (methods for altering amino acid            content of proteins), U.S. Pat. No. 5,850,016 (alteration of            amino acid compositions in seeds), WO 98/20133 (proteins            with enhanced levels of essential amino acids), U.S. Pat.            No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801            (high threonine), U.S. Pat. No. 6,664,445 (plant amino acid            biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increased            lysine and threonine), U.S. Pat. No. 6,441,274 (plant            tryptophan synthase beta subunit), U.S. Pat. No. 6,346,403            (methionine metabolic enzymes), U.S. Pat. No. 5,939,599            (high sulfur), U.S. Pat. No. 5,912,414 (increased            methionine), WO 98/56935 (plant amino acid biosynthetic            enzymes), WO 98/45458 (engineered seed protein having higher            percentage of essential amino acids), WO 98/42831 (increased            lysine), U.S. Pat. No. 5,633,436 (increasing sulfur amino            acid content), U.S. Pat. No. 5,559,223 (synthetic storage            proteins with defined structure containing programmable            levels of essential amino acids for improvement of the            nutritional value of plants), WO 96/01905 (increased            threonine), WO 95/15392 (increased lysine), U.S. Pat. Nos.            6,930,225, 7,179,955, U.S. Patent Application No.            2004/0068767, U.S. Pat. No. 6,803,498, WO 01/79516, and WO            00/09706 (Ces A: cellulose synthase), U.S. Pat. No.            6,194,638 (hemicellulose), U.S. Pat. No. 6,399,859 and U.S.            Pat. No. 7,098,381 (UDPGdH), U.S. Pat. No. 6,194,638 (RGP),            each of which is incorporated herein by reference in its            entirety.    -   4. Genes that Control Male Sterility:        -   There are several methods of conferring genetic male            sterility available, such as multiple mutant genes at            separate locations within the genome that confer male            sterility, as disclosed in U.S. Pat. Nos. 4,654,465 and            4,727,219 to Brar, et al. and chromosomal translocations as            described by Patterson in U.S. Pat. Nos. 3,861,709 and            3,710,511, each of which is incorporated herein by reference            in its entirety. In addition to these methods, Albertsen, et            al., U.S. Pat. No. 5,432,068, incorporated herein by            reference in its entirety, describe a system of nuclear male            sterility which includes: identifying a gene which is            critical to male fertility; silencing this native gene which            is critical to male fertility; removing the native promoter            from the essential male fertility gene and replacing it with            an inducible promoter; inserting this genetically engineered            gene back into the plant; and thus creating a plant that is            male sterile because the inducible promoter is not “on”            resulting in the male fertility gene not being transcribed.            Fertility is restored by inducing, or turning “on”, the            promoter, which in turn allows the gene that confers male            fertility to be transcribed.        -   A. Introduction of a deacetylase gene under the control of a            tapetum-specific promoter and with the application of the            chemical N-Ac-PPT. See PCT Application No. WO 01/29237,            incorporated herein by reference in its entirety.        -   B. Introduction of various stamen-specific promoters. See            PCT Application Nos. WO 92/13956 and WO 92/13957, each of            which is incorporated herein by reference in its entirety.        -   C. Introduction of the barnase and the barstar genes. See            Paul, et al., Plant Mol. Biol. 19:611-622, 1992).        -   For additional examples of nuclear male and female sterility            systems and genes, see U.S. Pat. Nos. 5,859,341; 6,297,426;            5,478,369; 5,824,524; 5,850,014; and 6,265,640, each of            which is incorporated herein by reference in its entirety.    -   5. Genes that Create a Site for Site-Specific DNA Integration:        -   This includes the introduction of FRT sites that may be used            in the FLP/FRT system and/or Lox sites that may be used in            the Cre/Loxp system. For example, see Lyznik, et al.,            Site-Specific Recombination for Genetic Engineering in            Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821,            each of which is incorporated herein by reference in its            entirety. Other systems that may be used include the Gin            recombinase of phage Mu (Maeser, et al., 1991; Vicki            Chandler, The Maize Handbook ch. 118, Springer-Verlag,            1994), the Pin recombinase of E. coli (Enomoto, et al.,            1983), and the R/RS system of the pSR1 plasmid (Araki, et            al., 1992).    -   6. Genes that Affect Abiotic Stress Resistance:        -   Genes that affect abiotic stress resistance (including but            not limited to flowering, pod and seed development,            enhancement of nitrogen utilization efficiency, altered            nitrogen responsiveness, drought resistance or tolerance,            cold resistance or tolerance, and salt resistance or            tolerance) and increased yield under stress. For example,            see WO 00/73475 where water use efficiency is altered            through alteration of malate; U.S. Pat. Nos. 5,892,009,            5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446,            6,706,866, 6,717,034, 6,801,104, international patent            publications WO 2000/060089, WO 2001/026459, WO 2001/035725,            WO 2001/034726, WO 2001/035727, WO 2001/036444, WO            2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430,            WO 2002/077185, WO 2002/079403, WO 2003/013227, WO            2003/013228, WO 2003/014327, WO 2004/031349, WO 2004/076638,            WO 98/09521, and WO 99/38977, each of which is incorporated            herein by reference in its entirety, describing genes,            including CBF genes and transcription factors effective in            mitigating the negative effects of freezing, high salinity,            and drought on plants, as well as conferring other positive            effects on plant phenotype; U.S. Patent Application No.            2004/0148654 and international publication WO 01/36596, each            of which is incorporated herein by reference in its            entirety, where abscisic acid is altered in plants resulting            in improved plant phenotype such as increased yield and/or            increased tolerance to abiotic stress; WO 2000/006341, WO            04/090143, U.S. Pat. Nos. 7,531,723 and 6,992,237, each of            which is incorporated herein by reference in its entirety,            where cytokinin expression is modified resulting in plants            with increased stress tolerance, such as drought tolerance,            and/or increased yield. Also see WO 02/02776, WO            2003/052063, JP2002281975, U.S. Pat. No. 6,084,153, WO            01/64898, U.S. Pat. Nos. 6,177,275 and 6,107,547            (enhancement of nitrogen utilization and altered nitrogen            responsiveness). For ethylene alteration, see U.S.            Application Nos. 2004/0128719, 2003/0166197 and WO            2000/32761, each of which is incorporated herein by            reference in its entirety. For plant transcription factors            or transcriptional regulators of abiotic stress, see e.g.            U.S. Application No. 2004/0098764 or U.S. Application No.            2004/0078852, each of which is incorporated herein by            reference in its entirety.        -   Other genes and transcription factors that affect plant            growth and agronomic traits such as yield, flowering, plant            growth and/or plant structure, can be introduced or            introgressed into plants, see e.g. WO 97/49811 (LHY), WO            98/56918 (ESD4), WO 97/10339 and U.S. Pat. Nos. 6,573,430            (TFL), 6,713,663 (FT), 6,794,560, 6,307,126 (GAI), WO            96/14414 (CON), WO 96/38560, WO 01/21822 (VRN1), WO 00/44918            (VRN2), WO 99/49064 (GI), WO 00/46358 (FR1), WO 97/29123, WO            99/09174 (D8 and Rht), and WO 2004/076638 and WO 2004/031349            (transcription factors), each of which is incorporated            herein by reference in its entirety.

Methods For Canola Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber, et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

-   -   A. Agrobacterium-Mediated Transformation—One method for        introducing an expression vector into plants is based on the        natural transformation system of Agrobacterium. See, for        example, Horsch, et al., Science 227:1229 (1985). A. tumefaciens        and A. rhizogenes are plant pathogenic soil bacteria which        genetically transform plant cells. The Ti and Ri plasmids of A.        tumefaciens and A. rhizogenes, respectively, carry genes        responsible for genetic transformation of the plant. See, for        example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).        Descriptions of Agrobacterium vector systems and methods for        Agrobacterium-mediated gene transfer are provided by Gruber, et        al., supra, Miki, et al., supra, and Moloney, et al., Plant Cell        Reports 8:238 (1989). See also, U.S. Pat. No. 5,563,055        (Townsend and Thomas), incorporated herein by reference in its        entirety.    -   B. Direct Gene Transfer—Several methods of plant transformation,        collectively referred to as direct gene transfer, have been        developed as an alternative to Agrobacterium-mediated        transformation. A generally applicable method of plant        transformation is microprojectile-mediated transformation        wherein DNA is carried on the surface of microprojectiles        measuring 1 to 4 μm. The expression vector is introduced into        plant tissues with a biolistic device that accelerates the        microprojectiles to speeds of 300 to 600 m/s which is sufficient        to penetrate plant cell walls and membranes. Sanford, et al.,        Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech.        6:299 (1988), Klein, et al., Bio/Technology 6:559-563 (1988),        Sanford, J. C., Physiol Plant 7:206 (1990), Klein, et al.,        Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580        (Christou, et al.) and U.S. Pat. No. 5,322,783 (Tomes, et al.),        each of which is incorporated herein by reference in its        entirety.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology 9:996 (1991).

Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985), Christou, et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine has also been reported. Hain, etal., Mol. Gen. Genet. 199:161 (1985) and Draper, et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Donn, et al., In Abstracts ofVIIth International Congress on Plant Cell and Tissue Culture IAPTC,A2-38, p 53 (1990); D'Halluin, et al., Plant Cell 4:1495-1505 (1992) andSpencer, et al., Plant Mol. Biol. 24:51-61 (1994).

Following transformation of canola target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well-known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular canola line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well-known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such as RFLP,Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), SSRs which are also referred toas Microsatellites, and SNPs. For exemplary methodologies, see Glick, etal., 1993. Methods in Plant Molecular Biology and Biotechnology. CRCPress, Boca Raton.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forSCV453784.

In addition to being used for identification of canola line SCV453784and plant parts and plant cells of line SCV453784, the genetic profilemay be used to identify a canola plant produced through the use ofSCV453784 or to verify a pedigree for progeny plants produced throughthe use of SCV453784. The genetic marker profile is also useful inbreeding and developing backcross conversions.

The present invention comprises a canola plant characterized bymolecular and physiological data obtained from the representative sampleof said variety deposited with the American Type Culture Collection(ATCC). Further provided by the invention is a canola plant formed bythe combination of the disclosed canola plant or plant cell with anothercanola plant or cell and comprising the homozygous alleles of thevariety.

Means of performing genetic marker profiles using SSR polymorphisms arewell-known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, byPCR, thereby eliminating the need for labor-intensive Southernhybridization. The PCR detection is done by use of two oligonucleotideprimers flanking the polymorphic segment of repetitive DNA. Repeatedcycles of heat denaturation of the DNA followed by annealing of theprimers to their complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase, comprise themajor part of the methodology. For example, see Batley, J., et al. 2007.Mol. Ecol. Notes (OnlineEarly Articles) and Plieske, J., et al., 2001.Theor. Appl. Genet. 102:689-694.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

The SSR profile of canola plant SCV453784 can be used to identify plantscomprising SCV453784 as a parent, since such plants will comprise thesame homozygous alleles as SCV453784. Because the canola variety isessentially homozygous at all relevant loci, most loci should have onlyone type of allele present. In contrast, a genetic marker profile of anF₁ progeny should be the sum of those parents, e.g., if one parent washomozygous for allele x at a particular locus, and the other parenthomozygous for allele y at that locus, then the F₁ progeny will be xy(heterozygous) at that locus. Subsequent generations of progeny producedby selection and breeding are expected to be of genotype x (homozygous),y (homozygous), or xy (heterozygous) for that locus position. When theF₁ plant is selfed or sibbed for successive filial generations, thelocus should be either x or y for that position.

In addition, plants and plant parts substantially benefiting from theuse of SCV453784 in their development, such as SCV453784 comprising abackcross conversion, transgene, or genetic sterility factor, may beidentified by having a molecular marker profile with a high percentidentity to SCV453784. Such a percent identity might be 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SCV453784.

The SSR profile of SCV453784 also can be used to identify essentiallyderived varieties and other progeny varieties developed from the use ofSCV453784, as well as cells and other plant parts thereof. Such plantsmay be developed using the markers identified in WO 00/31964, U.S. Pat.No. 6,162,967 and U.S. Pat. No. 7,288,386, each of which is incorporatedherein by reference in its entirety. Progeny plants and plant partsproduced using SCV453784 may be identified by having a molecular markerprofile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% geneticcontribution from canola variety, as measured by either percent identityor percent similarity. Such progeny may be further characterized asbeing within a pedigree distance of SCV453784, such as within 1, 2, 3,4, or 5 or less cross-pollinations to a canola plant other thanSCV453784 or a plant that has SCV453784 as a progenitor. Uniquemolecular profiles may be identified with other molecular tools such asSNPs and RFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such plant. Such unique SSR profiles mayarise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, an F₁ progeny produced from such variety, and progenyproduced from such variety.

Single-Gene Conversions

When the term “canola plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term “single gene converted plant,” as used herein, refersto those canola plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3,4, 5, 6, 7, 8 or more times to the recurrent parent. The parental canolaplant that contributes the gene for the desired characteristic is termedthe nonrecurrent or donor parent. This terminology refers to the factthat the nonrecurrent parent is used one time in the backcross protocoland therefore does not recur. The parental canola plant to which thegene or genes from the nonrecurrent parent are transferred is known asthe recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper, 1994; Fehr, Principles ofCultivar Development (1987)). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the single gene of interestto be transferred. The resulting progeny from this cross are thencrossed again to the recurrent parent and the process is repeated untila canola plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include but are not limited to,male sterility, waxy starch, herbicide tolerance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185; 5,973,234 and 5,977,445, each of which is incorporatedherein by reference in its entirety.

Introduction of a New Trait or Locus into SCV453784

Line SCV453784 represents a new base genetic variety into which a newlocus or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Backcross Conversions of SCV453784

A backcross conversion of SCV453784 occurs when DNA sequences areintroduced through backcrossing with SCV453784 utilized as the recurrentparent. Both naturally occurring and transgenic DNA sequences may beintroduced through backcrossing techniques. A backcross conversion mayproduce a plant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, or at least 5 crosses. Molecular marker assisted breeding orselection may be utilized to reduce the number of backcrosses necessaryto achieve the backcross conversion. For example, see Openshaw, S. J.,et al., Marker-assisted Selection in Backcross Breeding. In: ProceedingsSymposium of the Analysis of Molecular Data, August 1994, Crop ScienceSociety of America, Corvallis, Oreg., where it is demonstrated that abackcross conversion can be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage (seeHanauer, et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through backcrossconversion include, but are not limited to, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, altered fatty acid profile, alteredseed amino acid levels, altered seed oil levels, low phytate, industrialenhancements, disease resistance (bacterial, fungal or viral), insectresistance and herbicide tolerance. In addition, an introgression siteitself, such as an FRT site, Lox site or other site specific integrationsite, may be inserted by backcrossing and utilized for direct insertionof one or more genes of interest into a specific plant variety. In someembodiments of the invention, the number of loci that may be backcrossedinto SCV453784 is at least 1, 2, 3, 4, or 5 and/or no more than 6, 5, 4,3, or 2. A single locus may contain several transgenes, such as atransgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide tolerance. The gene forherbicide tolerance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of site specific integrationsystem allows for the integration of multiple genes at the convertedloci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,P. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant and easily recognized traits.

One process for adding or modifying a trait or locus in canola lineSCV453784 comprises crossing SCV453784 plants grown from SCV453784 seedwith plants of another canola variety that comprise the desired trait orlocus, selecting F₁ progeny plants that comprise the desired trait orlocus to produce selected F₁ progeny plants, crossing the selectedprogeny plants with the SCV453784 plants to produce backcross progenyplants, selecting for backcross progeny plants that have the desiredtrait or locus and the morphological characteristics of canola lineSCV453784 to produce selected backcross progeny plants; and backcrossingto SCV453784 three or more times in succession to produce selectedfourth or higher backcross progeny plants that comprise said trait orlocus. The modified SCV453784 may be further characterized as havingessentially all of the physiological and morphological characteristicsof canola line SCV453784 listed in Table 1 and/or may be characterizedby percent similarity or identity to SCV453784 as determined by SSRmarkers. The above method may be utilized with fewer backcrosses inappropriate situations, such as when the donor parent is highly relatedor markers are used in the selection step. Desired traits that may beused include those nucleic acids known in the art, some of which arelisted herein, that will affect traits through nucleic acid expressionor inhibition. Desired loci include the introgression of FRT, Lox andother sites for site specific integration, which may also affect adesired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny canola seed byadding a step at the end of the process that comprises crossingSCV453784 with the introgressed trait or locus with a different canolaplant and harvesting the resultant first generation progeny canola seed.

Tissue Culture of Canola

Further production of the SCV453784 line can occur by tissue culture andregeneration. Tissue culture of various tissues of canola andregeneration of plants therefrom is known and widely published. Forexample, reference may be had to Chuong, et al., “A Simple CultureMethod for Brassica Hypocotyl Protoplasts”, Plant Cell Reports 4:4-6(1985); Barsby, T. L., et al., “A Rapid and Efficient AlternativeProcedure for the Regeneration of Plants from Hypocotyl Protoplasts ofBrassica napus”, Plant Cell Reports, (Spring, 1996); Kartha, K., et al.,“In vitro Plant Formation from Stem Explants of Rape”, Physiol. Plant,31:217-220 (1974); Narasimhulu, S., et al., “Species Specific ShootRegeneration Response of Cotyledonary Explants of Brassicas”, Plant CellReports, (Spring 1988); Swanson, E., “Microspore Culture in Brassica”,Methods in Molecular Biology, Vol. 6, Chapter 17, p. 159 (1990). Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce canola plants having the physiological andmorphological characteristics of canola line SCV453784.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and pistils. Means for preparing andmaintaining plant tissue culture are well-known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234 and 5,977,445,describe certain techniques, each of which is incorporated herein byreference in its entirety.

Using SCV453784 to Develop Other Canola Varieties

Canola varieties such as SCV453784 are typically developed for use inseed and grain production. However, canola varieties such as SCV453784also provide a source of breeding material that may be used to developnew canola varieties. Plant breeding techniques known in the art andused in a canola plant breeding program include, but are not limited to,recurrent selection, mass selection, bulk selection, mass selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of canolavarieties in a plant breeding program requires, in general, thedevelopment and evaluation of homozygous varieties. For a generaldescription of rapeseed and canola breeding, see Downey, et al., 1987.Rapeseed and Mustard in Fehr, W. R. (ed.), Principles of CultivarDevelopment p. 437-486. N.Y., Macmillan and Co.; Thompson, K. F., 1983.Breeding winter oilseed rape Brassica napus. Adv. Appl. Bio. 7:1-104;and Ward, et al., 1985. Oilseed Rape. Farming Press Ltd., WharefedaleRoad, Ipswich, Suffolk.

Additional Breeding Methods

This invention, in an embodiment, is directed to methods for producing acanola plant by crossing a first parent canola plant with a secondparent canola plant wherein either the first or second parent canolaplant is line SCV453784. The other parent may be any other canola plant,such as a canola plant that is part of a synthetic or naturalpopulation. Any such methods using canola line SCV453784 are part ofthis invention: selfing, sibbing, backcrosses, mass selection, pedigreebreeding, bulk selection, hybrid production, or crosses to populations.These methods are well-known in the art and some of the more commonlyused breeding methods are described below. Descriptions of breedingmethods can be found in one of several reference books (e.g., Allard,Principles of Plant Breeding, 1960; Simmonds, Principles of CropImprovement, 1979; Sneep, et al., 1979).

The following describes breeding methods that may be used with canolaline SCV453784 in the development of further canola plants. One suchembodiment is a method for developing a line SCV453784 progeny canolaplant in a canola plant breeding program comprising: obtaining thecanola plant, or a part thereof, of line SCV453784 utilizing said plantor plant part as a source of breeding material and selecting a canolaline SCV453784 progeny plant with molecular markers in common with lineSCV453784 and/or with morphological and/or physiological characteristicsselected from the characteristics listed in Table 1. Breeding steps thatmay be used in the canola plant breeding program include pedigreebreeding, backcrossing, mutation breeding, and recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example SSR markers)and the making of double haploids may be utilized.

Another method involves producing a population of canola line SCV453784progeny canola plants, comprising crossing line SCV453784 with anothercanola plant, thereby producing a population of canola plants, which, onaverage, derive 50% of their alleles from canola line SCV453784. A plantof this population may be selected and repeatedly selfed or sibbed witha canola line resulting from these successive filial generations. Oneembodiment of this invention is the canola line produced by this methodand that has obtained at least 50% of its alleles from canola lineSCV453784.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus, the invention includes, in anembodiment, canola line SCV453784 progeny canola plants comprising acombination of at least two line SCV453784 traits selected from thegroup consisting of those listed in Table 1. In anther embodiment, theline SCV453784 comprises any combination of traits described herein. Ineither embodiment, the progeny canola plant may not be significantlydifferent for said traits than canola line SCV453784 as determined atthe 5% significance level when grown in the same environmentalconditions. Using techniques described herein, molecular markers may beused to identify said progeny plant as a canola line SCV453784 progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and the traits may then be measured onplants grown under the same environmental conditions. Once such avariety is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of canola line SCV453784 may also be characterized through theirfilial relationship with canola line SCV453784, as for example, beingwithin a certain number of breeding crosses of canola line SCV453784. Abreeding cross is a cross made to introduce new genetics into theprogeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween canola line SCV453784 and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4 or 5breeding crosses of canola line SCV453784.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asSCV453784 and another canola variety having one or more desirablecharacteristics that is lacking or which complements SCV453784. If thetwo original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations theheterozygous condition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. The developedvariety may comprise homozygous alleles at about 95% or more of itsloci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent but at the same time retain manycomponents of the non-recurrent parent by stopping the backcrossing atan early stage and proceeding with selfing and selection. For example, acanola variety may be crossed with another variety to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC1 or BC2.Progeny are selfed and selected so that the newly developed variety hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the non-recurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new canolavarieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of canola line SCV453784, comprising the steps ofcrossing a plant of canola line SCV453784 with a donor plant comprisinga desired trait, selecting an F₁ progeny plant comprising the desiredtrait, and backcrossing the selected F₁ progeny plant to a plant ofcanola line SCV453784. This method may further comprise the step ofobtaining a molecular marker profile of canola line SCV453784 and usingthe molecular marker profile to select for a progeny plant with thedesired trait and the molecular marker profile of SCV453784. In oneembodiment, the desired trait is a mutant gene or transgene present inthe donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. SCV453784 is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny and selfedprogeny. The selected progeny are cross pollinated with each other toform progeny for another population. This population is planted andagain superior plants are selected to cross pollinate with each other.Recurrent selection is a cyclical process and therefore can be repeatedas many times as desired. The objective of recurrent selection is toimprove the traits of a population. The improved population can then beused as a source of breeding material to obtain new varieties forcommercial or breeding use, including the production of a syntheticline. A synthetic line is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self pollination, directed pollinationcould be used as part of the breeding program.

Mutation Breeding

Mutation breeding is another method of introducing new traits intocanola line SCV453784. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (from 2500 to 2900 nm), or chemical mutagens (such as baseanalogues (5-bromo-uracil), related compounds (8-ethoxy caffeine),antibiotics (streptonigrin), alkylating agents (sulfur mustards,nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates,sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines.Once a desired trait is observed through mutagenesis the trait may thenbe incorporated into existing germplasm by traditional breedingtechniques. Details of mutation breeding can be found in Fehr, 1993.Principles of Cultivar Development, Macmillan Publishing Company. Inaddition, mutations created in other canola plants may be used toproduce a backcross conversion of canola line SCV453784 that comprisessuch mutation.

Breeding with Molecular Markers

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing canola line SCV453784. One use of molecularmarkers is QTL mapping. QTL mapping is the use of markers, which areknown to be closely linked to alleles that have measurable effects on aquantitative trait. Selection in the breeding process is based upon theaccumulation of markers linked to the positive effecting alleles and/orthe elimination of the markers linked to the negative effecting allelesfrom the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a canola plant for which canola line SCV453784 is a parent canbe used to produce double haploid plants. Double haploids are producedby the doubling of a set of chromosomes (1 N) from a heterozygous plantto produce a completely homozygous individual. For example, see Wan, etal., (1989) “Efficient Production of Doubled Haploid Plants ThroughColchicine Treatment of Anther-Derived Maize Callus”, Theor. Appl.Genet., 77:889-892 and U.S. Pat. No. 7,135,615, incorporated herein byreference in its entirety. This can be advantageous because the processomits the generations of selfing needed to obtain a homozygous plantfrom a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. Methods for obtaining haploidplants are also disclosed in Kobayashi, M., et al., J. Heredity71(1):9-14, 1980, Pollacsek, M., Agronomie (Paris) 12(3):247-251, 1992;Cho-Un-Haing, et al., J Plant Biol., 1996, 39(3):185-188; Verdoodt, L.,et al., February 1998, 96(2):294-300; Genetic Manipulation in PlantBreeding, Proceedings International Symposium Organized by EUCARPIA,Sep. 8-13,1985, Berlin, Germany; Chalyk, et al., 1994, Maize Genet Coop.Newsletter 68:47; Chalyk, S.

Thus, an embodiment of this invention is a process for making asubstantially homozygous SCV453784 progeny plant by producing orobtaining a seed from the cross of SCV453784 and another canola plantand applying double haploid methods to the F₁ seed or F₁ plant or to anysuccessive filial generation. Based on studies in maize and currentlybeing conducted in canola, such methods would decrease the number ofgenerations required to produce a variety with similar genetics orcharacteristics to SCV453784. See Bernardo, R. and Kahler, A. L., Theor.Appl. Genet. 102:986-992, 2001.

In particular, a process of making seed retaining the molecular markerprofile of canola line SCV453784 is contemplated, such processcomprising obtaining or producing F₁ seed for which canola lineSCV453784 is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of canola line SCV453784, and selecting progeny thatretain the molecular marker profile of SCV453784.

A pollination control system and effective transfer of pollen from oneparent to the other offers improved plant breeding and an effectivemethod for producing hybrid canola seed and plants. For example, theogura cytoplasmic male sterility (cms) system, developed via protoplastfusion between radish (Raphanus sativus) and rapeseed (Brassica napus)is one of the most frequently used methods of hybrid production. Itprovides stable expression of the male sterility trait (Ogura 1968),Pelletier, et al. (1983) and an effective nuclear restorer gene (Heyn1976).

In developing improved new Brassica hybrid varieties, breeders useself-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear malesterile (NMS) Brassica plants as the female parent. In using theseplants, breeders are attempting to improve the efficiency of seedproduction and the quality of the F₁ hybrids and to reduce the breedingcosts. When hybridization is conducted without using SI, CMS or NMSplants, it is more difficult to obtain and isolate the desired traits inthe progeny (F₁ generation) because the parents are capable ofundergoing both cross-pollination and self-pollination. If one of theparents is a SI, CMS or NMS plant that is incapable of producing pollen,only cross pollination will occur. By eliminating the pollen of oneparental variety in a cross, a plant breeder is assured of obtaininghybrid seed of uniform quality, provided that the parents are of uniformquality and the breeder conducts a single cross.

In one instance, production of F₁ hybrids includes crossing a CMSBrassica female parent, with a pollen producing male Brassica parent. Toreproduce effectively, however, the male parent of the F₁ hybrid musthave a fertility restorer gene (Rf gene). The presence of an Rf genemeans that the F₁ generation will not be completely or partiallysterile, so that either self-pollination or cross pollination may occur.Self pollination of the F₁ generation to produce several subsequentgenerations is important to ensure that a desired trait is heritable andstable and that a new variety has been isolated.

An example of a Brassica plant which is cytoplasmic male sterile andused for breeding is ogura (OGU) cytoplasmic male sterile (R.Pellan-Delourme, et al., 1987). A fertility restorer for oguracytoplasmic male sterile plants has been transferred from Raphanussativus (radish) to Brassica by Instit. National de Recherche Agricole(INRA) in Rennes, France (Pelletier, et al., 1987). The restorer gene,Rf1 originating from radish, is described in WO 92/05251 and inDelourme, et al., (1991). Improved versions of this restorer have beendeveloped. For example, see WO 98/27806 “Oilseed brassica containing animproved fertility restorer gene for ogura cytoplasmic male sterility”.

Other sources and refinements of CMS sterility in canola include thePolima cytoplasmic male sterile plant, as well as those of U.S. Pat. No.5,789,566, “DNA sequence imparting cytoplasmic male sterility,mitochondrial genome, nuclear genome, mitochondria and plant containingsaid sequence and process for the preparation of hybrids”; U.S. Pat. No.5,973,233 “Cytoplasmic male sterility system production canola hybrids”;and WO 97/02737 “Cytoplasmic male sterility system producing canolahybrids”; EP patent application 0 599042A “Methods for introducing afertility restorer gene and for producing F₁ hybrids of Brassica plantsthereby”; U.S. Pat. No. 6,229,072 “Cytoplasmic male sterility systemproduction canola hybrids”; U.S. Pat. No. 4,658,085 “Hybridization usingcytoplasmic male sterility, cytoplasmic herbicide tolerance, andherbicide tolerance from nuclear genes”; each of which is incorporatedherein by reference in its entirety.

Further, as a result of the advances in sterility systems, lines aredeveloped that can be used as an open pollinated variety (i.e. apureline line sold to the grower for planting) and/or as a sterileinbred (female) used in the production of F₁ hybrid seed. In the lattercase, favorable combining ability with a restorer (male) would bedesirable. The resulting hybrid seed would then be sold to the growerfor planting.

The development of a canola hybrid in a canola plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, although different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in canola, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid between a defined pair of inbredswill always be the same. Once the inbreds that give a superior hybridhave been identified, the hybrid seed can be reproduced indefinitely aslong as the homogeneity of the inbred parents is maintained.

Combining ability of a line, as well as the performance of the line, isa factor in the selection of improved canola lines that may be used asinbreds. Combining ability refers to a lines contribution as a parentwhen crossed with other lines to form hybrids. The hybrids formed forthe purpose of selecting superior lines are designated test crosses. Oneway of measuring combining ability is by using breeding values. Breedingvalues are based on the overall mean of a number of test crosses. Thismean is then adjusted to remove environmental effects and it is adjustedfor known genetic relationships among the lines.

Hybrid seed production requires inactivation of pollen produced by thefemale parent. Incomplete inactivation of the pollen provides thepotential for self-pollination. This inadvertently self-pollinated seedmay be unintentionally harvested and packaged with hybrid seed.Similarly, because the male parent is grown next to the female parent inthe field there is also the potential that the male selfed seed could beunintentionally harvested and packaged with the hybrid seed. Once theseed from the hybrid bag is planted, it is possible to identify andselect these self-pollinated plants. These self-pollinated plants willbe genetically equivalent to one of the inbred lines used to produce thehybrid. Though the possibility of inbreds being included in hybrid seedbags exists, the occurrence is rare because much care is taken to avoidsuch inclusions. These self-pollinated plants can be identified andselected by one skilled in the art, either through visual or molecularmethods.

Brassica napus canola plants, absent the use of sterility systems, arerecognized to commonly be self-fertile with approximately 70 to 90percent of the seed normally forming as the result of self-pollination.The percentage of cross pollination may be further enhanced whenpopulations of recognized insect pollinators at a given growing site aregreater. Thus open pollination is often used in commercial canolaproduction. Descriptions of other breeding methods that are commonlyused for different traits and crops can be found in one of severalreference books (e.g., Allard, 1960; Simmonds, 1979; Sneep, et al.,1979; Fehr, 1987).

INDUSTRIAL USES

Currently Brassica napus canola is recognized as an increasinglyimportant oilseed crop and a source of meal in many parts of the world.The oil as removed from the seeds commonly contains a lesserconcentration of endogenously formed saturated fatty acids than othervegetable oils and is well suited for use in the production of salad oilor other food products or in cooking or frying applications. The oilalso finds utility in industrial applications. Additionally, the mealcomponent of the seeds can be used as a nutritious protein concentratefor livestock.

Canola oil has the lowest level of saturated fatty acids of allvegetable oils. “Canola” refers to rapeseed (Brassica) which has aerucic acid (C_(22:1)) content of at most 2 percent by weight based onthe total fatty acid content of a seed, and which produces, aftercrushing, an air-dried meal containing less than 30 micromoles (μmol)per gram of defatted (oil-free) meal. These types of rapeseed aredistinguished by their edibility in comparison to more traditionalvarieties of the species.

Canola line SCV453784 can be used in the production of an ediblevegetable oil or other food products in accordance with knowntechniques. The solid meal component derived from seeds can be used as anutritious livestock feed. Parts of the plant not used for human oranimal food can be used for biofuel.

TABLES

In Table 2, selected oil quality characteristics of the seed of canolaline SCV453784 are compared with oil quality characteristics of the samecanola lines referenced in Table 1. The data in Table 2 includes resultson seed samples collected from 20 testing locations and are presented asaverages of the values observed. Column 1 shows the variety, column 2shows the percent saturated fatty acid content, column 3 shows thepercent oleic acid content, column 4 shows the percent linoleic acidcontent and column 5 shows the percent linolenic acid content.

Compared to the two canola lines SCV378221 and SCV204738, the averagespresented in Table 2 indicate that seed of canola line SCV453784 of thepresent invention has a percent saturated fatty acid content in a normalrange, a similar average percent oleic acid content, a lower averagepercent linoleic acid and a percent protein linolenic acid that is inbetween.

TABLE 2 Oil Quality Characteristics of SCV453784 Compared to TwoProprietary Canola Lines 2 3 4 5 1 Sat. Fatty Oleic Acid Linoleic AcidLinolenic Acid Variety Acids % % % % SCV453784 7.80% 66.62% 17.08% 6.63%SCV378221 7.61% 64.17% 19.28% 7.09% SCV204738 7.49% 66.93% 17.41% 6.43%

In Table 3, selected characteristics of a single cross hybrid G88115containing canola line SCV453784 are compared with characteristics oftwo commercial canola check varieties. Trials data values are shown forG88115 and the average of the two commercial canola varieties, Q2 and46A65, with the values shown being representative of data collected froma specified number of trial locations (“No. Locs”) over all zones andyears tested. Column 1 shows the variety, column 2 shows the yield as apercent of the average of the check varieties, column 3 shows the plantlodging ratings (on a scale of 1 to 5, see Definitions), column 4 showsthe days to maturity, column 5 shows the percent saturated fatty acidcontent, column 6 shows the plant height, column 7 shows the percentglucosinolate content in micromoles, column 8 shows the percent oilcontent, column 9 shows the percent protein content, column 10 shows theresistance rating to blackleg disease, and column 11 shows theresistance rating to Fusarium wilt disease.

Compared to the average of the values recorded for Check Q2 and Check46A45, the hybrid (G88115) containing SCV453784 of the present inventionhas higher yield, a comparable lodging rating, a days to maturity ratingthat is slightly earlier, a comparable percent saturated fat content, ashorter plant height, a lower glucosinolate content, a higher percentoil content, and a slightly lower percent protein content, withresistance to blackleg and Fusarium wilt diseases.

TABLE 3 Characteristics of a Hybrid Containing SCV453784 Compared to TwoCommercial Varieties* 2 3 4 5 6 7 8 9 10 11 1 Yield Lodging DMat SatsHeight Gluc Oil Prot BL FW Var. % rating Days % cm μm/g % % ratingrating Q2 99.8 1.2 106.4 7.00 102.9 13.37 46.87 45.22 MR R 46A65 100.21.5 105.9 6.69 102.4 14.72 47.15 46.83 MR R AVG of 100 1.3 106.1 6.85102.7 14.04 47.01 46.02 MR R CHECKS G88115 127.5 1.4 105.1 6.86 100.98.00 50.14 45.39 R R No. Loc 34 34 34 34 34 34 34 34 3 3 *Hybrid G88115compared to commercial varieties Q2 and 46A65. Note: Q2 and 46A65 areused as check varieties in the official Canadian variety registrationtrials conducted by the Western Canada Canola/Rapeseed RecommendingCommittee, Inc. Data shown for each variety and characteristic are themean values over all zones and years tested.

DEPOSIT INFORMATION

A deposit of the Monsanto Canada Inc. proprietary canola line designatedSCV453784 disclosed above and recited in the appended claims has beenmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was Jan. 28, 2010.The deposit of 2,500 seeds was taken from the same deposit maintained byMonsanto Canada Inc. since prior to the filing date of this application.All restrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. 1.801-1.809. TheATCC accession number is PTA-10627. The deposit will be maintained inthe depository for a period of 30 years, or 5 years after the lastrequest, or for the effective life of the patent, whichever is longer,and will be replaced as necessary during that period.

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, Internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Although embodiments of the invention have been described using specificterms, devices, and methods, such description is for illustrativepurposes only. The words used are words of description rather than oflimitation. It is to be understood that changes and variations may bemade by those of ordinary skill in the art without departing from thespirit or the scope of the present invention, which is set forth in thefollowing claims. In addition, it should be understood that aspects ofthe various embodiments may be interchanged both in whole or in part.Therefore, the spirit and scope of the appended claims should not belimited to the description of the versions contained therein.

What is claimed is:
 1. A seed of canola line SCV453784, a representativesample of seed of which was deposited under ATCC Accession No.PTA-10627.
 2. A canola plant, or a part thereof, produced by growing theseed of claim
 1. 3. A tissue culture produced from protoplasts or cellsfrom the plant of claim 2, wherein said cells or protoplasts of thetissue culture are produced from a plant part selected from the groupconsisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell, root, root tip, anther, pistil, flower, shoot, stem, petiole andpod.
 4. A canola plant regenerated from the tissue culture of claim 3,wherein the plant has essentially all of the morphological andphysiological characteristics of line SCV453784, as shown in Table
 1. 5.A method for producing a canola seed comprising crossing two canolaplants and harvesting the resultant canola seed, wherein at least one ofthe two canola plants is the canola plant of claim
 2. 6. A canola seedproduced by the method of claim
 5. 7. A canola plant, or a part thereof,produced by growing said seed of claim
 6. 8. A method for producing amale sterile canola plant wherein the method comprises transforming thecanola plant of claim 2 with a nucleic acid molecule that confers malesterility.
 9. A male sterile canola plant produced by the method ofclaim
 8. 10. A method of producing an herbicide tolerant canola plantwherein the method comprises transforming the canola plant of claim 2with a transgene that confers herbicide tolerance to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, 2,4-D, Dicamba, L-phosphinothricin, triazine,hydroxyphenylpyruvate dioxygenase inhibitor, protoporphyrinogen oxidaseinhibitor, phenoxy proprionic acid, cyclohexone, and benzonitrile. 11.An herbicide tolerant canola plant produced by the method of claim 10.12. A method of producing an insect- or pest-resistant canola plantwherein the method comprises transforming the canola plant of claim 2with a transgene that confers insect or pest resistance.
 13. An insect-or pest-resistant canola plant produced by the method of claim
 12. 14.The canola plant of claim 13, wherein the transgene encodes a Bacillusthuringiensis endotoxin.
 15. A method of producing a disease resistantcanola plant wherein the method comprises transforming the canola plantof claim 2 with a transgene that confers disease resistance.
 16. Adisease resistant canola plant produced by the method of claim
 15. 17. Amethod of producing a canola plant with modified fatty acid metabolismor modified carbohydrate metabolism wherein the method comprisestransforming the canola plant of claim 2 with a transgene encoding aprotein selected from the group consisting of fructosyltransferase,levansucrase, alpha-amylase, invertase, and starch branching enzyme orencoding an antisense of stearyl-ACP desaturase.
 18. A canola planthaving modified fatty acid metabolism or modified carbohydratemetabolism produced by the method of claim
 17. 19. Meal prepared fromthe seed of the canola line of claim
 1. 20. An unblended canola oilextracted from the seed of the canola line of claim
 1. 21. A method ofintroducing a desired trait into canola line SCV453784 wherein themethod comprises: (a) crossing a SCV453784 plant, wherein arepresentative sample of seed was deposited under ATCC Accession No.PTA-10627, with a plant of another canola line that comprises a desiredtrait to produce progeny plants, wherein the desired trait is selectedfrom the group consisting of male sterility, herbicide tolerance, insectresistance, pest resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, modified seed yield, modified oil percent,modified protein percent, modified lodging resistance, and resistance tobacterial disease, fungal disease, or viral disease; (b) selecting oneor more progeny plants that have the desired trait to produce selectedprogeny plants; (c) crossing the selected progeny plants with theSCV453784 plants to produce backcross progeny plants; (d) selecting forbackcross progeny plants that have the desired trait and essentially allof the physiological and morphological characteristics of canola lineSCV453784 listed in Table 1; and (e) repeating the crossing the selectedprogeny step and selecting for backcross progeny step two or more timesto produce selected third or higher backcross progeny plants thatcomprise the desired trait and essentially all of the physiological andmorphological characteristics of canola line SCV453784 as shown inTable
 1. 22. A canola plant produced by the method of claim 21, whereinthe plant has the desired trait.
 23. The canola plant of claim 22,wherein the desired trait is herbicide tolerance and the resistance isconferred to an herbicide selected from the group consisting ofimidazolinone, sulfonylurea, glyphosate, glufosinate, 2,4-D, Dicamba,L-phosphinothricin, triazine, hydroxyphenylpyruvate dioxygenaseinhibitor, protoporphyrinogen oxidase inhibitor, phenoxy proprionicacid, cyclohexone and benzonitrile.
 24. The canola plant of claim 22,wherein the desired trait is insect resistance and the insect resistanceis conferred by a transgene encoding a Bacillus thuringiensis endotoxin.25. The canola plant of claim 22, wherein the desired trait is modifiedfatty acid metabolism or modified carbohydrate metabolism and saiddesired trait is conferred by a nucleic acid encoding a protein selectedfrom the group consisting of phytase, fructosyltransferase,levansucrase, α-amylase, invertase and starch branching enzyme orencoding an antisense of stearyl-ACP desaturase.
 26. A method ofproducing a male sterile canola seed wherein the method comprisescrossing the canola plant of claim 2 with a male sterile canola plantand harvesting the resultant seed.